Date of Award

8-13-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Giovanni Gadda

Second Advisor

Markus W. Germann

Third Advisor

Jun Yin

Fourth Advisor

Samer Gozem

Abstract

Propionate 3-nitronate (P3N) is a natural toxin that irreversibly inhibits mitochondrial succinate dehydrogenase. P3N poisoning leads to a variety of neurological disorders and even death. Nitronate monooxygenase (NMO) from Cyberlindnera saturnus (CsNMO) and Pseudomonas aeruginosa PAO1 (PaNMO) serve as paradigms for Class I NMO, which catalyze the oxidation of P3N involving single electron transfer. In this dissertation, the crystallographic structure of CsNMO was solved and demonstrated a highly conserved three-dimensional structure and active site with respect to NMO from PaNMO. The role of conserved residues in the active site of Class I NMO, e.g. Y109, Y254, Y299, Y303, and K307 in PaNMO in substrate binding and catalysis were investigated using site-directed mutagenesis, steady-state kinetics and pH effects on the UV-visible absorption spectrum. The study revealed that a protonated tyrosine is required for binding of the negatively charged P3N substrate. We also report that PaNMO can stabilize both the neutral and anionic semiquinones anaerobically for hours, providing a constant protein environment to study their photochemical and photophysical properties.

Choline oxidase catalyzes two-step oxidation of choline to glycine betaine with betaine aldehyde as an intermediate. The FAD cofactor is covalently attached to the choline oxidase via H99 through an 8α-N3-histidyl linkage. In the active site of choline oxidase, S101 and H466 are located on two extent loops, ~ 4 Å from the flavin C4a atom. In this dissertation, a charge-induced, reversible C4a-S-cysteinyl-8α-N3-histidyl FAD was engineered by replacing S101 with a cysteine. The mechanistic rationale for the stabilization of de novo C4a-S-cysteinyl-flavins was illustrated with rapid kinetics, pH, kinetic isotope effects and proton inventory. A photoinduced transient C4a-N-histidyl-8α-N3-histidyl FAD in choline oxidase wild-type was also observed with the aid of fluorescence excitation spectroscopy. Site-directed mutagenesis, solvent equilibrium isotope effects and pH effects on the stoke shifts of flavin in choline oxidase wild-type demonstrated H466 as the adduct on the C4a atom of flavin upon excitation, and provided a mechanistic rationale involving photoinduced electron transfer (PET) for the formation of the novel photoinduced transient flavin C4a adduct.

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